Alkaline phosphatase from Microorganism
|Description:||Alkaline phosphatase (E.C.126.96.36.199)|
|Reaction :||Orthophosphoric-monoester + H₂O → alcohol + Orthophosphate|
|Origin :||Microorganism (Bacterium)|
|Specific activity :||≧6,000 U/mg-protein (37°C・pH=9.8, pNPP is used as a substrate)|
|Molecular weight :||Approx. 100,000 (by gel filtration)|
|Optimum pH :||Approx. 9.8|
|pH stability :||6.4 – 9.0 (25°C, 24hr)|
|Thermalstability :||≦60°C (pH7.5,1hr)|
|Available substrates:||p-Nitrophenyl phosphate, Dioxietane-based substrates, Acridan-based
substrates, 5-Bromo-4-Chloro-3-Indoxyl Phosphate (BCIP),
4-Methylumbelliferyl Phosphate, etc.
|Stability :||Stable at 4°|
3. The data in ELISA test
4. Alkaline phosphatase ASSAY
The appearance of p-Nitrophenol is measured at 405nm by spectrophotometry .
One unit causes the formation of one micromole of p-Nitrophenol per minute under the conditions described below .
|A. Diethanolamine buffer:||1M [Dilute 9.66ml of diethanolamine (MW=105.14) in 60ml H₂O, add 5ml of 0.1M MgCl₂ and, after adjusting the pH at 37°C to 9.8 with 2N HCl, fill up to 100ml with H₂O]|
|B. pNPP solution:||0.674M [2.5g p-Nitorphenylphosphate disodium salt (MW=371.16) / 10ml Diethanolamine buffer (A)] (Prepare freshly)|
|C. Enzyme diluent:||30mM Triethanolamine, 1mM MgCl₂ , 0.1mM ZnCl₂. 0.1% Triton X-100 pH=7.6|
|Concentration in assay mixture|
1. Prepare the following working solution (30.5ml) in a brownish bottle and store on ice. (Prepare freshly)
30ml Diethanolamine buffer (A)
0.5ml pNPP solution (B)
2. Pipette 3.0ml of working solution into a cuvette (d=1.0cm) and equilibrate at 37°C for about 5minutes .
3. Add 0.1ml of the enzyme solution* and mix by gentle inversion.
4. Record the increase of optical density at 405nm against water for 3 to 5 minutes in a spectrophotometer
thermostated at 37°C, and calculate the ΔOD per minute from the initial linear portion of the curve (ΔODtest) . At the same time , measure the blank rate (ΔODblank) by the same method as test except that the enzyme diluent (C) is added instead of the enzyme solution .
* Dilute to 0.1 – 0.3 U/ml with ice cold enzyme diluent (C), immediately before assay .
Activity can be calculated by using the following formula :
ΔOD/min (ΔOD test-ΔOD blank)×Vt×df
Volume activity (U/ml) =
- : Total volume (3.1ml)
- : Sample volume (0.1ml)
- : Millimolar extinction coefficient of p-Nitrophenol under the assay conditions (㎠/micromole)
- : Light path length (cm)
- : Dilution factor